Our goal is the purification and biochemical characterization of the antitumor substance tumor necrosis factor (TNF), which is released into the serum of C. parvum inoculated mice, following the iv injection of endotoxin. TNF is defined as that activity which induces necrosis in Meth A tumors growing intradermally in BALB/c mice or which kills mouse L-cells (Clone 929) (in culture). To achieve this goal physical, chemical and immunological techniques will be employed. We plan to identify the tissue of origin, define the physiological conditions which govern the formation and release of TNF and determine if a physiological role for TNF exists. We have partially purified TNF by (NH4)2SO4 fractionation, sephadex gel filtration and polyacrylamide electrophoresis. TNF is an alpha2-globulin, glycoprotein in nature, has a molecular weight of 150,000 and it dissociates into four protein subunits during acrylamide electrophoresis. Since SDS treatment can dissociate these protein subunits still further without destroying the biological activity, it will be used in the further purification of TNF. TNF-like activity has been extracted from liver microsomes of normal mice and from CP-endotoxin treated mice. The identity of this activity with that of the serum factor has not been established. The specific activity of TNF from CP-endotoxin treated mouse liver is 10 times normal mouse serum. The activity of this material is approximately one tenth of that from serum of CP-endotoxin mice. TNF from normal animals is not endotoxic since concentrated preparations do not contain sufficient endotoxin (Limulus assay) to induce necrosis of Meth A tumors in vivo. The possibility of a physiological role for TNF is indicated by our finding that TNF strongly inhibits the in vitro activity of colony stimulating factor (CSF) and nerve growth factor (NGF). We anticipate that this program will be complete in three years.